This invention relates to the process for the preparation of cholesterol esterase and more specifically to the process for the preparation of cholesterol esterase by fermentation of a cholesterol esterase-producing microorganism of the genus Pseudomonas.
Cholesterol esterase (E C 3.1.1.13) is an enzyme which catalyzes the hydrolysis of cholesterol esters into cholesterol and fatty acids, and is known to be distributed widely in animals and plants.
Recently, it has been proposed to use cholesterol esterase in the determination of total serum cholesterol [Clinical Chemistry, Vol 20, No. 4 pages 470-475 (1974)]. The determination of total cholesterol in serum is important for diagnostic purposes in connection with arteriosclerosis, cerebral hemorrhage and hepatic disorders.
Cholesterol exists in the free form and ester form in serum. In the proposed determination method, a sample of serum is treated with cholesterol esterase and cholesterol oxidase. In this treatment, cholesterol esters in the sample are hydrolyzed into free cholesterol and fatty acids, and free form cholesterol in the serum and the liberated cholesterol are oxidized into cholestenone generating hydrogen peroxide. The total cholesterol can be determined by measuring oxygen uptake, cholestenone or hydrogen peroxide by a conventional method. The above method is simple and convenient and therefore is considered to be applicable to the practical assay.
As is already mentioned, cholesterol esterase is known to be widely distributed in natural sources. However, a development of a process of the commercial production of the enzyme is now in demand. The present inventors have carried out studies on the fermentative production of cholesterol esterase by microorganisms. The present inventors initially carried out the screening of cholesterol esterase producers from various bacteria based on the test on the utilization of cholesterol esters and subsequent test on the productivity of cholesterol esterase. As the result, the present inventors have found that good cholesterol esterase producers are found among microorganisms of the genus Pseudomonas, particularly, those of the species Pseudomonas fluorescens.